The present invention relates to the use of phenylalanine amide derivatives, and pharmaceutical compositions containing these compounds as Chemokine/CCR-3 receptor antagonists.
Chemokines are a superfamily of small secreted proteins. There are approximately 30 distinct chemokines known with many others being characterized. See Oppenheim et al., Properties of the Novel Proinflammatory Supergene xe2x80x9cIntercrinexe2x80x9d Cytokine Family, Ann. Rev. Immun., 9, 617-648 (1991); and Baggiolini, et al., Interleukin-8 and Related Chemotactic Cytokines-CXC and CC Chemokines, Adv. Immun., 55, 97-179 (1994). The properties of the chemokines suggest that they are essential for leukocyte trafficking and inflammatory processes, and are thus important components in a number of disease states. See Kita et al., Chemokines Active on Eosinophils: Potential Roles in Allergic Inflammation, J. Exp. Med., 183, 2421-2426 (1996); Strieter, et al., xe2x80x9cThe Good, the Bad and the Uglyxe2x80x9d The Role of Chemokines in Models of Human Diseases, J. Immun., 157, 3583-3586 (1996); and Baggiolini, Eotaxin: a VIC (Very Important Chemokine) of Allergic Inflammation, J. Clin. Invest., 97,587 (1996).
Chemokines mediate their effects via interactions with 7TM-G-protein coupled receptors on the surface of immune and inflammatory cells. Eosinophils are proinflammatory granulocytes that play a major role in allergic diseases, such as bronchial asthma, allergic rhinitis, pruritis and atopic dermatitis. Upon activation, eosinophils release lipid mediators, cytotoxic proteins, oxygen metabolites and cytokines, all of which have the potential to produce pathophysiology. Numerous studies have demonstrated the presence of eosinophils or eosinophil-specific products in inflamed tissues in human diseases.
The mechanisms responsible for the selective infiltration of eosinophils in allergic diseases have yet to be clarified. Recently, a CC chemokine, eotaxin, was identified in guinea pigs and demonstrated to be present in a guinea pig model of allergic airway inflammation. See Jose, et al., Eotaxin: A Potent Eosinophil Chemoattractant Cytokine Detected in Guinea Pig Model of Allergic Airways Inflammation, J. Exp. Med., 179, 881-887 (1994); and Jose, et al., Eotaxin: Cloning of an Eosinophil Chemoattractant Cytokine and Increased mRNA Expression in Allergen-challenged Guinea-pig Lungs, Biochem. Biophys. Res. Comm., 205, 788-794 (1994). The human homologue of Guinea-pig eotaxin has been expressed and has been shown to induce eosinophil infiltration when injected into the skin of the rhesus monkey. See Ponath, et al., Cloning of the Human Eosinophil Chemoattractant, Eotaxin: Expression, Receptor Binding, and Functional Properties Suggest a Mechanism for Selective Recruitment of Eosinophils, J. Clin. Invest., 97, 604-612 (1996).
The cloning, expression and characterization of a novel Cxe2x80x94C chemokine receptor, designated CCR-3 from peripheral blood eosinophils and from an eosinophil cDNA library have also been reported. See Kitaura, et al., Molecular Cloning of Human Eotaxin, an Eosinophil-selective CC Chemokine, and Identification of a Specific Eosinophil Eotaxin Receptor, CC Chemokine Receptor 3, J. Biol. Chem., 271, 7725-7730 (1996); Ahuja, et al., Cloning and Functional Expression of a Human Eosinophil CC Chemokine Receptor, J. Biol. Chem., 270, 16491-16494 (1995); Daugherty, et al., Cloning, Expression and Characterization of the Human Eosinophil Eotaxin Receptor, J. Exp. Med. 183,,2349-2354 (1996); and Ponath, et al., Molecular Cloning and Characterization of a Human Eotaxin Receptor Expressed Selectively on Eosinophils, J. Exp. Med., 183, 2437-2448 (1996).
Eotaxin, MCP-4 and, to a lesser extent, RANTES and MCP-3 activate this receptor. The CCR-3 receptor is expressed at high levels on eosinophils; typically 40,000-400,000 receptors per cell are present. This is 10-100 fold more than the other chemokine receptor (CCR-1) expressed in eosinophils. Monoclonal antibodies raised to the CCR-3 receptor demonstrate that the receptor is primarily restricted to eosinophils and a subset of Th2 T-cells. This restricted expression on eosinophils and T-cells may be responsible for the selective recruitment of eosinophils and Th2 T-cells in allergic inflammation. Additionally, CCR-3 is potently activated by eotaxin 1, eotaxin and MCP-4. See Stellato et al., Production of the Novel CC Chemokine MCP-4 by Airway Cells and Comparison of Its Biological Activity to other CC-Chemokines. J. Clin. Invest. 99, 92-936 (1997). In contrast, other known chemokines appear to activate more than one chemokine receptor, e.g. RANTES binds to CCR-1, CCR-3, CCR-4 and CCR-5 receptors.
The foregoing research advances have provided the impetus to investigate the inhibition of eosinophil-specific chemokines in order to examine its role in blocking cellular infiltration in inflamed tissues. CCR-3 receptor antagonists thus offer a unique approach toward decreasing the pathophysiology associated with allergic diseases. Antagonism of this receptor may be useful in the treatment of allergic disorders, including but not limited to bronchial asthma, allergic rhinitis, eczema, nasal polyposis, conjunctivitis, atopic dermatitis, inflammatory bowel disorder and pruritis.
The present invention involves phenylalanine amide derivatives represented by Formula (1) hereinbelow and their use as CCR-3 receptor antagonists which is useful in the treatment of a variety of diseases associated with allergic disorders, including but not limited to bronchial asthma, allergic rhinitis, nasal polyposis, atopic dermatitis and pruritis.
The present invention further provides methods for antagonizing CCR-3 receptors in an animal, including humans, which comprises administering to a subject in need of treatment an effective amount of a compound of Formula (I) or (II) as indicated hereinbelow.
The compounds useful in the present methods are selected from Formula (I) or (II) hereinbelow: 
wherein
n is an integer from 0 to 3;
R1 is selected from the group consisting of N-carbobenzoxy-L-Phe, N-Ac-L-Pro, C1-6 alkyl, OC1-4 alkyl, aryl and heteroaryl, unsubstituted, monosubstituted, disubstituted or trisubstituted, with any substituents being independently selected from the group consisting of C1-4 alkyl, COaryl, OCH2O, NO2, Cl and OCH3.
R2 represents C1-4 alkyl or benzyl;
m is an integer from 1 to 3: and
R3 is independently selected from the group consisting of OH, OC1-4 alkyl, NO2, NH2, halo, naphthyl, and OCOphenyl; or 
xe2x80x83wherein R represents indolylmethyl, phenyl or (CH2)2phenyl.
Preferably, in Formula(I), n represents 1
Preferably, in Formula(I), at the R1 position, alkyl represents cC6H11 or n-Bu. Preferably, O-alkyl represents O-t-Bu. Preferably, aryl represents phenyl or naphthyl. Preferably, heteroaryl represents thienyl, furyl, pyridyl, or quinolinyl.
Preferably, substituents at R1 are independently selected from the group consisting of C1-2 alkyl, CO-phenyl, OCH2O, NO2, Cl, OH, and OCH3.
Preferably, at R2, alkyl moieties are methyl or ethyl.
A preferred halo moiety at R3 is iodo.
Preferably, in Formula (II), the carbon atom marked with the asterix (*) represents an S configuration.
Preferred compounds of the present invention include:
(S)-Ethyl-2-(1-napthoylamino)3-(4-nitrophenyl)propionate,
(S)-Isopropyl-2-(1-napthoylamino)3-(4-nitrophenyl)propionate,
(S)-Methyl-2-(1-napthoylamino)3-(4-nitrophenyl)propionate,
(S)-Benzyl-2-(1-napthoylamino)3-(4-nitrophenyl)propionate,
(S)Ethyl-2-(1-napthoylamino)3-(4-chlorophenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(4-hydroxyphenyl)propionate,
(S,S)-Ethyl-2-(2-benzyloxycarbonylamino-3-phenylpropionylamino)-3-(4-hydroxyphenyl)propionate,
(S,S)-Ethyl-2-(N-acetylpyrrolidine-2-benzoylamino)-3-(4-hydroxy-phenyl)propionate,
(S)-Ethyl-2-cyclohexanylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(3,3-diphenylpropionylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(3-phenylpropionylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-[2-(2-naphthyl)acetylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(4-phenylbutyrylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-pentanylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-pentanylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(4-benzoylbenzoylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(2-furanyl)amino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(1-naphthoylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(5-hydroxyindonyl)amino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-piperonylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-picolinylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(3-nitro-4-chlorobenzoylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(3-hydroxy-4-nitrobenzoylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(8-quinolinylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-phenylpropionate,
(S)-Methyl-2-benzoylamino-3-(4-hydroxyphenyl)propionate,
(S)-Benzyl-2-benzoylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(4-methoxyphenyl)propionate,
(S)-Ethyl-2-tert-butyloxycarbonylamino-3-(4-nitrophenyl)propionate,
(S)-Ethyl-2-tert-butyloxycarbonylamino-3-(4-aminophenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(3,5-diiodo-4-hydroxyphenyl)propionate,
(S)-Ethyl-2-carboxybenzoylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(1-naphthyl)propionate,
(xc2x1)-Ethyl-2-benzolyamino-3-[3-(benzoyloxy)phenyl]propionate,
(xc2x1)-Ethyl-2-benzoylamino-3-(3-hydroxyphenyl)propionate,
(R,S)-Ethyl-2-benzoylamino-3-(2-hydroxylphenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(4-aminophenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(4-nitrophenyl)propionate,
(S)-Ethyl-2-(2-phenylacetylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(3-indoyl)propionate,
(xc2x1)-Ethyl-2-(benzoylamino)-2-phenylacetate, and
(xc2x1)-Ethyl-2-(benzoylamino)-4-phenylbutyrate.
More preferred compounds of the present invention include:
(S)-Ethyl-2-(1-napthoylamino)3-(4-nitrophenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-cyclohexanylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-(1-naphthoylamino)-3-(4-hydroxyphenyl)propionate,
(S)-Benzyl-2-benzoylamino-3-(4-hydroxyphenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(4-methoxyphenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(1-naphthyl)propionate,
(xc2x1)-Ethyl-2-benzolyamino-3-[3-(benzoyloxy)phenyl]propionate,
(xc2x1)-Ethyl-2-benzoylamino-3-(3-hydroxyphenyl)propionate,
(R,S)-Ethyl-2-benzoylamino-3-(2-hydroxylphenyl)propionate,
(S)-Ethyl-2-benzoylamino-3-(4-nitrophenyl)propionate,
(xc2x1)-Ethyl-2-(benzoylamino)-2-phenylacetate, and
(xc2x1)-Ethyl-2-(benzoylamino)-4-phenylbutyrate.
The most preferred compounds useful in the present invention include:
(S)-Ethyl-2-(1-naphthoylamino)-3-(4hydroxyphenyl)propionate,
(S)-Ethyl-2-(1-napthoylamino)3-(4-nitrophenyl)propionate, and
(S)-Ethyl-2-benzoylamino-3-(4-nitrophenyl)propionate.
Also included in the present invention are pharmaceutically acceptable salt complexes. Preferred are the ethylene diamine, sodium, potassium, calcium and ethanolamine salts. The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All of these compounds and diastereomers are contemplated to be within the scope of the present invention.
The present compounds can be prepared by the using the overall strategies provided hereinbelow. Such strategies are readily found in the art. See e.g. Comprehensive Organic Transformations, R. C. Larock, VCH Publishers, 1989(and references therein); and Organic chemistry, Vol. 1; I. L. Finar, Longman Group, 1973.
The present compounds are readily prepared by conventional acylation methods (for example as used in peptide synthesis) and well known to those skilled in the art and are exemplified in Scheme I below: 
A: EtOH, HCl, reflux; B: PhCO2H, EDCI, HOBt, NMM, HOBt or PHCOCl, Et3N, CH4Cl2 
The amino acids of Formula 1, Scheme 1 are either commercially available (eg. Aldrich Chemical Co., Milwaukee, Wis.) or may be prepared from commercially available amino acids by appropriate functional group manipulations by standard methods known to those skilled in the art.
With appropriate manipulation and protection of any chemical functionality, synthesis of the remaining compounds of the present invention is accomplished by methods analogous to those above and to those described in the Experimental section.
In order to use a compound of the present invention or a pharmaceutically acceptable salt thereof for the treatment of humans and other mammals it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
As used herein, xe2x80x9ctreatmentxe2x80x9d of a disease includes, but is not limited to prevention, retardation and prophylaxis of the disease.
The present compounds are useful for the treatment of diseases including but not limited to bronchial asthma, allergic rhinitis, nasal polyposis, eczema, conjunctivitis, atopic dermatitis, pruritis and inflammatory bowel disease.
Compounds of the present invention and their pharmaceutically acceptable salts may be administered in a standard manner for the treatment of the indicated diseases, for example orally, parenterally, sub-lingually, dermally, transdermally, rectally, via inhalation or via buccal administration.
Compositions of the present compounds and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules, creams and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil, olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils, and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.
A typical suppository formulation comprises a present compound or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats or their synthetic analogs.
Typical dermal and transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer a single dose.
Each dosage unit for oral administration contains suitably from 0.1 mg to 500 mg/Kg, and preferably from 1mg to 100 mg/Kg, and each dosage unit for parenteral administration contains suitably from 0.1 mg to 100 mg/Kg, of a present compound or a pharmaceutically acceptable salt thereof calculated as the free acid. Each dosage unit for intranasal administration contains suitably 1-400 mg and preferably 10 to 200 mg per person. A topical formulation contains suitably 0.01 to 5.0% of a present compound. The daily dosage regimen for oral administration is suitably about 0.01 mg/Kg to 40 mg/Kg, of a present compound or a pharmaceutically acceptable salt thereof calculated as the free acid, the daily dosage regimen for parenteral administration is suitably about 0.001 mg/Kg to 40 mg/Kg, of a present compound or a pharmaceutically acceptable salt thereof calculated as the free acid, the daily dosage regimen for intranasal administration and oral inhalation is suitably about 10 to about 500 mg/person. The active ingredient may be administered from 1 to 6 times a day, sufficient to exhibit the desired activity.
No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention.
The biological activity of the present compounds is demonstrated by the following tests:
Human eosinophils were purified by standard CD16 cell depletion using a Miltenyi cell separation column and a magnetic Super Macs magnet. Eosinophils which were  greater than 95% pure as assessed by DiffQuick staining and light microscopy were washed in PBS and resuspended in binding buffer (RPMI-1640+25 mM Hepes+0.1% Gelatin+0.1% sodium azide+0.008% CHAPS). Into a 96 well plate (Dynatek) 200,000 eosinophils, 0.25 nM 125I-Eotaxin (Amersham Plc), and compound of interest (1 nM to 100 uM) was added. This mixture of cells compound and ligand was allowed to incubate for 60 min at room temperature before harvesting. For harvesting, free ligand from bound ligand was separated over a Packard Unifilter-96 GFC, (cat #6005174) which had been pre-blocked with 1% polyethylenimine (Sigma Cat #P3143) and 1% Bovine Serum Albumin (BSA) for 2 hours prior to use. After drying, and sealing the plate with Topseal (Packard Topseal A Cat #6005185) 50 ul of MicroScint (Packard Microscint-20 Cat #6013621) was added to each well. Bound from free 125I-eotaxin was separated using a Packard Filtermate 196, 96-well plate harvester. To determine total and non-specific binding (NSB) three wells for each condition were set aside. For total binding and NSB, wells received all additions except compound. In addition NSB wells received 200 nM cold eotaxin (PeproTech, Rocky Hill, N.J.). Radioactivity associated with the filter was assessed in a Packard Top-count Microplate Scintillation Counter model number 49872V. Percent control binding was assessed by first subtracting the NSB from each well and then expressing the number of counts (CPM) associated with the compound treated sample as a percent of the control binding in the absence of compound addition.
Biological Assay for the Determination of the Inhibition of Intracellular Calcium Mobilization by Compounds of the Present Invention
RBL-2H3 cells expressing the human CCR-3 receptor were grown in cell medium (EMEM medium with Earl""s salts) containing 2mM L-Glutamine, 0.4 mg/ml G418 Sulfate from GIBCO BRL and 10% heat inactivated fetal calf serum from Hyclone Laboratories. The cells were seeded 30,000 cells,/well into 96-well black clear bottom sterile plates from Costar. The seeded plate was incubated overnight at 37xc2x0 C. in 5% CO2. On the day of the assay the cell medium was aspirated before addition of calcium dye loading solution consisting of: 1 mg/mL bovine serum albumin (BSA), 1.5 mM sulfinpyrazone from SIGMA and 4 uM Fluo-3 AM dye from Molecular Probes in cell medium, thereafter the 96-well plate was incubated for 1 hour at 37xc2x0 C. The loading solution containing dye was then aspirated and replaced with fresh solution without dye after which the plate was incubated for a further 10 mins at 37xc2x0 C.
This solution was aspirated and cells were washed with assay buffer (Kreb""s Ringer Henseleit pH 7.4 containing 1 mM CaCl2, 1.1 mM MgCl2, 1.5 mM sulfinpyrazone and 1.0 mg/mL Gelatin) after aspirating the wash, 100 uLs of fresh assay buffer was added to all the wells and the plate was incubated for five minutes at 37xc2x0 C. before transferring to the Fluorescent Imaging Plate Reader (FLIPR) instrument. The assay and data acquisition were initiated by addition of 50 uLs of sample diluted to a relevant concentration in assay buffer. After 2 mins 75 uLs of human Eotaxin, from PeproTech Inc., diluted to an appropriate concentration in assay buffer with 1 mg/mL BSA (no gelatin) was added to the plate and data was acquired fro an additional 1.5. mins. Concentration response data for compounds showing inhibition of calcium mobilization were performed in the presence of 33 nM Eotaxin to obtain the IC50 values. IC50 is the concentration ofd compound needed to inhibit 50% of the Eotaxin response.
Animal Model for the in vivo Evaluation of CCR-3 Antagonists
(Gonzalo, J. A. et al, Immunity, 1996, 4, 1.)
BALs were obtained from Guinea Pigs (xc2x1compound) 24 h after ovalbumin (OA) exposure to eotaxin administered via inhalation. The animals were euthanized by cervical dislocation and exsanguinated. The lungs were lavaged with 50 ml of DulBecco""s PBS (5xc3x9710 cc), which was aspirated after a gentle chest massage. The BAL fluid was spun down and the pellet was resuspended in 0.25% NaCl to lyse residual erythrocytes. After centrifugation, the pellet was resuspended again in 0.9% NaCl. After a total cell count, slides were prepared and stained. The cells were differentiated into eosinophils, neutrophils and monocytes by counting a minimum of 200 cells and expressing the results as a percentage of total cells.
Alternatively, OA sensitized Guinea Pigs (xc2x1compound) were exposed to OA via inhalation 24 h after OA exposure and lungs were obtained as described above and assessed for eosinophil infltration.